Poplar Extrafloral Nectaries: Two Types, Two Strategies of
Indirect Defenses against Herbivores1[C][W]
María Escalante-Pérez 2, Mario Jaborsky 2, Silke Lautner, Jörg Fromm, Tobias Müller, Marcus Dittrich,
Maritta Kunert, Wilhelm Boland, Rainer Hedrich, and Peter Ache*
University Würzburg, Biozentrum, Julius-von-Sachs-Institut für Biowissenschaften, D–97082 Wuerzburg,
Germany (M.E.-P., M.J., R.H., P.A.); University Hamburg, Zentrum Holzwirtschaft, D–21031 Hamburg,
Germany (S.L., J.F.); University Würzburg, Bioinformatics Department, Am Hubland/Biozentrum, D–97074
Wuerzburg, Germany (T.M., M.D.); Max Planck Institute for Chemical Ecology, 07745 Jena, Germany (M.K.,
W.B.); and Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia (R.H.)
Many plant species grow extrafloral nectaries and produce nectar to attract carnivore arthropods as defenders against
herbivores. Two nectary types that evolved with Populus trichocarpa (Ptr) and Populus tremula 3 Populus tremuloides (Ptt) were
studied from their ecology down to the genes and molecules. Both nectary types strongly differ in morphology, nectar
composition and mode of secretion, and defense strategy. In Ptt, nectaries represent constitutive organs with continuous
merocrine nectar flow, nectary appearance, nectar production, and flow. In contrast, Ptr nectaries were found to be holocrine
and inducible. Neither mechanical wounding nor the application of jasmonic acid, but infestation by sucking insects, induced Ptr
nectar secretion. Thus, nectaries of Ptr and Ptt seem to answer the same threat by the use of different mechanisms.
Plants secrete nectar to achieve two highly important mutualistic interactions with animals: pollination
and indirect defense (Brandenburg et al., 2009; Heil,
2011). Floral nectar is secreted within the flowers and
serves pollination. Extrafloral nectar (EFN) is secreted
in general on the vegetative parts and attracts members of the third trophic level as a means of indirect
defense against herbivores (Heil, 2008). In fact, EFN is
one of the very few antiherbivore defense mechanisms
for which an effect on plant fitness has been demonstrated unambiguously in a number of field studies
(Chamberlain and Holland, 2009). Nectar secretion
mechanisms have been intensively studied at the
phenotypic level, and we now know that plants can
adjust nectar quantity and quality to several biotic
factors, such as ontogenetic stage, consumer identity,
consumption rate, and, in the case of EFN, current leaf
damage (Heil, 2011). However, the biochemical,
physiological, and genetic mechanisms that underlie
1
This work was supported by the Deutsch Forschungsgemeinschaft
(grant nos. SFB567, TP C7, and AC97/4–2 P5) within the German joint
research group Poplar: A Model to Address Tree-Specific Questions.
2
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Peter Ache ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.196014
1176
the regulation of nectar secretion remain widely unknown.
For more than 100 years, scientists have discussed
two alternative mechanisms for the secretion of floral
nectar. However, even less is known about EFN secretion (Escalante-Pérez and Heil, 2012). Holocrine
secretion is characterized by programmed cell death
that, in a one-step process, causes release of the entire
cell content into exterior parts. In this case, the nectar is
produced and kept within the cells until the plasma
membrane is ruptured (Vesprini et al., 2008). This type
of secretion has so far not been described for extrafloral
nectaries. By contrast, merocrine secretion is associated
with living nectar-secreting cells by prolonged largescale exocytosis. Concerning merocrine secretion, it is
debated whether the “prenectar,” after uploading from
the phloem, moves via an apoplastic pathway or via
a vesicle-based symplastic pathway (Vassilyev, 2010;
Heil, 2011, and refs. therein). For the symplastic
pathway, transport via endocytosis and exocytosis,
molecular transport across the plasmalemma, and
transport via plasmodesmata have been considered
(Fahn, 1988b; Gaffal et al., 2007; Kram and Carter,
2009; Kram et al., 2009). The merocrine type of secretion may even use eccrine secretion, which comprises
carrier-based transport of individual molecules across
the cell membrane, or granulocrine secretion, relying
on transport of a fluid phase that seems to be governed
by endoplasmic reticulum- or dictyosome-derived vesicles. The latter membrane structures subsequently
fuse with the plasmalemma and finally are released
into the apoplast (Dauwalder and Whaley, 1982; Sauer
et al., 1994; Jürgens and Geldner, 2002). To study the
mechanisms that underlie the secretion of EFN, here
Plant PhysiologyÒ, July 2012, Vol. 159, pp. 1176–1191, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. All Rights Reserved.
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
we applied physiological assays, electron microscopy,
and transcriptomic analysis to gain new insight in this
rather complex matter. Thereby, the special focus was
on the relation between nectary type and defense.
Extrafloral nectaries of the genus Populus were first
described by Trelease (1881). Since that time, not much
attention has been paid to defense mechanisms used
by different poplar species (Wooley et al., 2007). The
genus Populus is a known host for many herbivorous
insects and pathogens. One member of the genus,
Populus trichocarpa (Ptr), has been sequenced, providing access to genome-wide expression studies (Tuskan
et al., 2006) in general and defense genes in particular.
Populus thus provides an excellent model tree to study
biotic stress management (Arimura et al., 2004; Lawrence
et al., 2006; Ralph et al., 2006).
Nectaries in general consist of three components:
epidermis with or without stomata or trichomes; specialized parenchyma that produces or stores nectar;
and a subnectary parenchyma composed of bigger
cells more loosely packed (Stpiczy
nska et al., 2005;
Kaczorowski et al., 2008). Detailed studies have shown
that decreases in herbivory rates associated with EFN
secretion result from mechanical leaf damage, jasmonic acid (JA) production, and/or volatile emission
(Heil et al., 2001; Linsenmair et al., 2001; Mathews
et al., 2007; Radhika et al., 2008). Volatile organic
compounds represent another indirect defense to herbivores (Arimura et al., 2000; Kessler and Baldwin,
2002; Gershenzon, 2007) and have also been demonstrated to be part of a “plant-to-plant communication”
network. Volatiles can act as alarm signals for neighboring plants that yet remain undamaged (Heil and
Silva Bueno, 2007; Kost and Heil, 2008). According to
the conventional view, nectar is mainly composed of
sugars and amino acids, originates from phloem sap,
and enters the secretory cells via plasmodesmataconnected parenchyma cells (Fahn, 1988a). However,
nectar might also contain substances generally not
carried in the phloem sap, such as inorganic ions,
proteins, lipids, organic acids, phenolics, and alkaloids
(Jones, 1983). Given the enormous variability in nectar
features regarding volume, concentration, and composition, the latter hypothesis appears to be a kind of
oversimplification (Pacini et al., 2003; Heil, 2011).
We have used two different species of Populus (Ptr
and Populus tremula 3 Populus tremuloides [Ptt]) to investigate the structure, nectar production, composition, and gene expression in their extrafloral nectaries.
The results of our multidisciplinary study demonstrate
that EFNs of the two poplar species studied differ in
their chemical composition and are secreted via a
holocrine mechanism in the first species and via merocrine secretion in the second species.
RESULTS
Two Types of Nectary Structures Harbor Unique
Secretion Systems
In the two poplar species Ptr and Ptt, pairs of nectaries are located on each side of the leaf blade near the
petiole (Fig. 1). Ptt nectaries were bigger, whereas
those of Ptr released more nectar (Fig. 1, C and F).
Light microscopy with Ptr nectaries documented the
structure described for many floral nectaries. They
contain an epidermis in addition to a nectar and a
subnectary parenchyma (Stpiczy
nska et al., 2005; Wist
and Davis, 2006; Kaczorowski et al., 2008; Wenzler
et al., 2008; Fig. 2A). Ptt nectaries develop a nectar and
subnectary parenchyma as well. In contrast to Ptr,
however, the outer layer consisted of modified epidermal
Figure 1. Positioning of poplar extrafloral nectaries. In both species, nectary pairs localize at the base of the
leaf blade. A to C, Ptr. D to F, Ptt. In the
leaf overviews (A and D), arrows denote nectary positions. In the leaf base
enlargements, nectaries in detail (arrows in B and E), Ptr release of large
nectar amounts (C), and large Ptt nectaries releasing small nectar amounts
(F) are shown.
Plant Physiol. Vol. 159, 2012
1177
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
Figure 2. Anatomy of Ptr and Ptt nectaries. A to F, Ptr nectary microscopy.
A, Overview. B, Detailed view of the
epidermal cell wall and cuticle (inset)
with microchannels marked by arrows.
C and D, Overview of nectary parenchyma cells with some extant thick
walls and symplastic connections
(square). E and F, Plasmodesmata between cells (arrows; E) and numerous
small vacuoles (F, left) and large nuclei
(F, right). G to L, Ptt nectary microscopy. G, Overview. H, Overview of
secretory cells. I, Plasmodesmata (arrows) between two secretory cells.
J and K, Secreted vesicles and multivesicular bodies (arrows) occur within
the epidermal cells and in the upper
apoplastic space. Note the partial
loosening of the cell wall. L, Symplastic connections between the secretory cells and the basolateral
neighbors are absent (black rectangles). ch, Chloroplast; CU, cuticle;
CW, cell wall; e, endoplasmic reticulum; m, mitochondria; mvb, multivesicular bodies; NE, nectary epidermis;
NP, nectary parenchyma; SNP, subnectary parenchyma; v, vacuole.
cells (Fig. 2G). In transmission electron microscopy
(TEM) analyses, we found the surface of nectary epidermal cells of Ptr covered by a cuticle entirely.
Therein, microchannels appeared as narrow tubular
interruptions in continuity with the cell wall. These
fibrillar outgrowths of the outer epidermal cell walls
might represent a path for passive nectar flow (Fig. 2B;
Supplemental Fig. S1). In Ptr, the isodiametric nectary
parenchyma cells (including the secretory cells) could
be distinguished from ground parenchyma by the
presence of a dense granular cytoplasm, rich in ribosomes, mitochondria, and chloroplasts (Fig. 2C). These
specialized features reflected the high metabolic activity required for nectar production. Nectary parenchyma cells generally grow thin walls (D’Amato,
1984). In contrast to Ptt, these cells possessed unusually
thick walls with numerous pits and associated plasmodesmata connecting the protoplasts of adjacent cells
(Fig. 2, D and E). Around the symplastic connections,
we could visualize numerous chloroplasts, plastids
containing plastoglobuli, mitochondria along with rough
endoplasmic reticulum, and dictyosomes (Golgi apparatus). The numerous vacuoles of the nectary parenchyma appeared small and surrounded by dark-stained
matter (Fig. 2).
With Ptt nectaries, surprisingly, in TEM and light
microscopy and after wax staining by Sudan III, no
cuticle was found covering the secretory epidermal
cells (Supplemental Fig. S2). Ultrastructural analyses
of Ptt nectaries showed that the secretory cells (Fig.
2H), representing specialized epidermal cells, were
interconnected at their lateral side by a large number
of plasmodesmata (Fig. 2I). Such connections were
absent between the nectary parenchyma at the basolateral side (Fig. 2L; Supplemental Fig. S1). In Ptt,
extrafloral nectary vesicles are located in the outer
apoplastic space as well as in the tip of the secretory
cells (Fig. 2, J and K; Supplemental Fig. S1). As with
1178
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
floral nectaries, these structures were associated with
epidermal cell endocytosis in the process of reabsorption of nonconsumed nectar (Nepi and Stpiczy
nska,
2007, 2008).
Ptt nectaries were treated with the membranestaining but membrane-impermeable fluorescent dye
FM4-64 (Fischer-Parton et al., 2000). The intracellular
presence of dye in longitudinal sections of FM4-64
stained nectaries strongly pointed to endocytosis events
(Supplemental Fig. S3). FM4-64, however, failed to cross
the apoplastic space between secretory cells and parenchymal neighbors.
Stress, Nectary Development, and Activity
In spring, many emerging Ptt and Ptr leaves harbor
nectaries (Fig. 3B). To test whether this phenomenon
results from heritable genetics, the extrafloral nectary
densities of field-grown Ptt trees were determined. The
populations of leaves with (38%) and without (62%)
nectaries were highly conserved among individual
trees (Fig. 3A). Similar data were obtained with Ptr
(Supplemental Fig. S4). These results are consistent
with the general effectiveness of nectaries against herbivore attack (Heil et al., 2001, 2005; Kost and Heil,
2008). Sixty percent (Ptr) or 80% (Ptt) of leaves carrying
nectaries developed no visible symptoms of herbivore
attack, 35% (Ptr) or 15% (Ptt) were slightly damaged,
and only 2% (both species) were severely affected (Fig.
3C; Supplemental Fig. S3B). Nectary-free leaves, however, exhibited a higher percentage of damage with Ptt
and Ptr (Fig. 3; Supplemental Fig. S4).
Nectaries appeared with the onset of leaf emergence
on both poplar species. In contrast to Ptt, where nectar
was secreted continuously over weeks, Ptr nectaries
released nectar within a few days only. Most of the
nectaries of the latter sort died after nectar release.
However, when the tree was specifically stressed after
the first nectar secretion, new nectaries occurred side
by side or on top of dead ones. These secondary nectaries showed the same secretion characteristics as the
initial population (Fig. 4).
So, how was the secondary nectar production of Ptr
induced? To test whether EFN production in Ptr results
from mechanical stress, leaves of Ptr were wounded by
puncturing the leaf blade with a needle (Fig. 5A). After
this procedure, however, nectar production was not
observed.
To test whether persistent wounding, a feeding fingerprint of herbivores, initiates nectar production, an
automated damage procedure was applied (Fig. 5, B
and C). The computer-controlled mechanical caterpillar MecWorm mimics the damage caused by herbivores in terms of the spatiotemporal pattern of leaf
destruction (Mithöfer et al., 2005). Even these nearnatural wounding settings did not trigger nectar production. This indicates that Ptr nectar production
seems not to be initiated simply by wounding of leaves
but requires another stimulus instead. The same result
Figure 3. Nectary density and effectiveness against herbivore attack.
A, Conserved percentage of Ptt leaves with and without nectaries. B,
Both Ptr (bottom row) and Ptt (top row) leaves emerging in spring
presented nectaries (arrows). C, Ptt extrafloral nectary effectiveness.
Leaves with nectaries appeared less damaged by herbivores. Values are
means 6 SE; n = 11 branches (759 leaves). [See online article for color
version of this figure.]
was obtained when the MecWorm-damaged tissue was
additionally treated with the oral secretion of the
Mediterranean climbing cutworm (Spodoptera littoralis).
Finally, caterpillars of S. littoralis, Spodoptera exigua, and
Lymantria dispar, which are polyphagous insects and
thus feed on poplar leaves, were placed on Ptr (Fig. 5D).
Again, neither nectary nor nectar production was observed within 48 h of caterpillar feeding. These results
clearly showed that herbivore-evoked leaf damage is
not causing Ptr nectar secretion. But when intact
plants of Ptr were subjected to mealy bugs (Hemiptera:
Pseudicoccidae), which, in contrast to caterpillars, feed
on phloem sap, nectar secretion set in (Fig. 5, E and F).
At the beginning of the experiment, less than 10% of
the leaves showed nectaries without nectar. Four days
after infestation, 42% of all leaves were equipped with
nectaries, and about 50% of them produced nectar
(data not shown). In most cases, the secondary nectar
production occurred upon attack by sucking insects
(Fig. 5, E–H). Thus, Ptr nectar formation seemed to be
confined to specific types of herbivores.
Plant Physiol. Vol. 159, 2012
1179
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
Figure 4. Ptr requires new nectaries for repeated nectar secretion. A,
First holocrine nectar secretion. B, Nectaries died after holocrine secretion. C, Secondary nectar-secreting nectaries on top of dead ones.
Volatiles Are Not Involved in Ptr Nectary Induction
Suc, Fru, and Glc in poplar EFN appeared relatively
constant in the different samples from the same species
but differed remarkably between species (Supplemental
Fig. S5). In Ptt, the ratios between Glc, Fru, and Suc
were equal, whereas, in contrast, the percentage of Suc
in Ptr nectar was rather low. Thus, EFNs of Ptt likely
attract generalists such as honeybees, wasps, and parasitic wasps, whereas those of Ptr attract more specialized visitors, such as ants (Supplemental Fig. S6; Steidle
and van Loon, 2003; Heil et al., 2005).
Amino acids are the second most common class of
solutes in nectar, and their composition is important for
nectar taste (Baker and Baker, 1983). Insects possess receptors that enable them to sense amino acids (Shiraishi
Volatile organic compounds (VOCs) in plants take
part in indirect defense to overcome herbivore attack.
They attract predatory arthropods and/or repel herbivores (Arimura et al., 2000; De Moraes et al., 2001;
Kessler and Baldwin, 2002; Gershenzon, 2007). Moreover, green leaf volatiles released from lima bean
(Phaseolus lunatus) leaves after herbivore damage are
able to induce EFN flow in undamaged neighboring
plants (Heil et al., 2008). Herbivory or even mechanical
leaf damage elevates endogenous levels of JA, stimulating volatile biosynthesis. As a consequence, externally added JA also triggered volatile emission from
plants (Boland et al., 1995) or induced nectar flow from
extrafloral nectaries of Macaranga tanarius (Heil et al.,
2001). Mechanical damage was obviously not sufficient
to induce Ptr secondary nectar release. Thus, the role of
volatiles in EFN secretion of this poplar species was
examined. To trigger the VOC emission of Ptr by
jasmonates or herbivory-related stressors, poplar leaves
were continuously damaged by the MecWorm or by S.
littoralis, S. exigua, and L. dispar caterpillars. After mechanical damage, herbivory, and application of JA or
coronalon, which acts as a mimic of jasmonoyl-Ile
(Schüler et al., 2001; Svoboda and Boland, 2010), VOCs
were released, but no nectaries were formed and
no nectar flow was stimulated. The emitted volatiles
comprised (E)-b-ocimene, 4,8-dimethyl-1,3,7-nonatriene,
farnesene, nerolidol, and 4,8,12-trimethyltridec-1,3,7,11tetraene (Danner et al., 2011). The compounds C10H16O
and C10H14 result from catalytic oxidation of the originally emitted (E)-b-ocimene by the active carbon trap
used for volatile collection (Sonwa et al., 2007). Irrespective of the ocimene artifacts, the compounds shown
in Figure 6 are characteristic for induced volatiles observed after induction with jasmonates or by herbivory.
Thus, we concluded that in Ptr, volatiles were induced as
expected, but nectar production was not VOC dependent.
Nectar Composition Feeds Back on a Visitor’s Attraction
Nectar is a complex mixture of metabolites. The
sweet nectar blend is dominated by Suc, Glc, and Fru.
Nectar with high Suc content attracts generalists,
whereas nectars with higher hexose contents are preferred by specialists (Heil et al., 2005). The amounts of
Figure 5. Induction of Ptr nectar release by sucking insects. A, Mechanical damage performed by a needle. B, MecWorm setup for
mimicking caterpillar-caused mechanical leaf damage. C, Continuous
leaf wounding by MecWorm. D, S. exigua feeding on Ptr leaves. E and
F, Mealy bugs on a leaf blade with ongoing nectar secretion. G, Aphids
on a leaf bottom side. H, Top side of the leaf shown in G with ongoing
nectar secretion.
1180
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
Figure 6. Volatiles emitted by Ptt after treatment with chemical elicitors and feeding larvae of S. littoralis. Control (crtl), n = 11; JA
(1 mM), n = 13; coronalon (Cor; 0.1 mM), n = 9; S. littoralis (Slit), n = 15. n-Bromodecane was used as an internal standard; C10H14 and
C10H16O are oxidation products of ocimene. DMNT, 4,8-Dimethyl-1,3,7-nonatriene; TMTT, 4,8,12-trimethyltridec-1,3,7,11-tetraene.
and Kuwabara, 1970). Interestingly, amino acids responsible for sweet taste, like Phe, Trp, and Val, were
well represented in both Ptt and Ptr nectar samples but
low or absent in leaves (Fig. 7; Supplemental Fig. S7).
Pro, which has a “salty taste,” was only found in nectar
samples but not in leaf extracts (Fig. 7; Supplemental Fig.
S7). In Ptt, Phe and Gln dominated, whereas in Ptr, Asn,
His, and Tyr were most abundant.
Nectary-Specific Genes
To identify the genes encoding defense proteins and
to gain a deeper insight into the Ptt nectary transcriptome, poplar DNA microarrays (Affymetrix) were
hybridized with RNA obtained from extrafloral nectaries or nectary-free leaf sections. With extrafloral
nectaries, only the apical fraction of the organ harboring the nectar-producing cells was sampled (Fig. 8,
inset). mRNA samples of nectaries with secretory cells
enriched and leaf sections without nectaries were analyzed. The 500 most differentially expressed genes
(Supplemental Table S1) were considered for further
analysis. Among them in nectaries, 365 (73%) appeared
up-regulated and 135 (27%) appeared down-regulated.
Array data were validated by quantitative real-time
PCR (qPCR) with a set of randomly selected transcripts and a set of further analyzed transcripts (see
below; Supplemental Table S2). For an unbiased view
Plant Physiol. Vol. 159, 2012
1181
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
nectaries. The strong induction of metabolic activity
clusters in this organ was already predicted by light
microscopy and TEM (Fig. 2). Genes related to hormone action and metabolism, lipids (vesicle transport),
as well as sugar metabolism and the cell wall appeared
up-regulated, in many cases to very high levels.
Exocytosis-Associated Genes
Figure 7. Most abundant amino acids in poplar EFN. A, Ptt. B, Ptr. Of
the aromatic amino acids, particularly the sweet Phe and Trp were
present in higher quantities. Pro was not detectable in leaf extracts of
both species. Values are means 6 SD; n = 4.
of the impact of differential expression on nectar biology, gene functions were analyzed by using MapMan (Usadel et al., 2009; Fig. 8). Among the 27
different gene clusters found, nine (signaling, stress,
transport, development, and carbohydrate, lipid, hormone, cell wall, and secondary metabolism) were
considered as important for nectary development. Interestingly, in the population of the 102 genes related
to these clusters, about 90% appeared up-regulated in
Microscopic inspection provided evidence for granulocrine secretion only with Ptt and not with Ptr nectaries. Recycling of lipids and proteins is characteristic
for this kind of secretion and a prerequisite for ongoing
nectar flow. Secretory vesicles thus appeared to be
very prominent in secretory cells from Ptt nectaries
(Fig. 2). Accordingly, in the full array data set, we
could identify at least 21 genes related to exocytosis
(Table I). Among them, five were remarkably higher
expressed in the nectaries (2- to 7.9-fold) and eight
were slightly induced (1.5- to 1.9-fold). In this population, seven nectary genes were linked to multivesicular body formation. Most of them belong to the
SNARE superfamily. One family member, PEP12
syntaxin, was found 4.6-fold enriched in nectary cells.
Six genes encoded proteins of the trans-Golgi network
(TGN), such as the RabA (four), RabE, and VHA-a1
class. We identified a number of genes that, according
to Zárský et al. (2009), seemed to be involved in
vesicle or TGN-to-plasma membrane carrier formation. Among them, three genes appeared remarkably
up-regulated. The latter species of Ptt nectaryexpressed genes belong to the SEC14, Ala-3, and
dynamin classes. SEC14 encodes a phosphatidylinositol transfer protein and Ala-3 a flippase. Both are essential for vesicle budding from the Golgi complex
(Sha et al., 1998; Litvak et al., 2005; Poulsen et al.,
2008), whereas dynamins seem to be required for
membrane scission (Bashkirov et al., 2008).
These granulocrine secretion-related transcripts
were then monitored by qPCR in samples of Ptr. With
this poplar species, none of the genes appeared to be
induced in nectaries (Table II), supporting the hypothesis of different secretion mechanisms used by Ptt
and Ptr extrafloral nectaries.
Figure 8. Clustering of Ptt differently expressed
genes (extrafloral nectaries versus leaves). The 500
highest differentially expressed genes were imported
into MapMan 3.1.1 and classified accordingly. The
inset shows the Ptt nectary, where only the upper
third (line) was used for RNA isolation. In the pie
chart, most of the genes were not annotated (not
assigned). Clusters related to nectary function are
highlighted, and MapMan rasters are included. The
boxes near the gene clusters represent the genes of
that particular category (blue squares = up-regulated,
red squares = down-regulated). Note that genes in
these clusters are almost all up-regulated. CHO,
Carbohydrate.
1182
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
Table I. Exocytosis-associated genes in Ptt nectaries
Public IDa
Arabidopsis Genome
Initiative Codeb
Annotationc
Fold Change
Log2 of
Fold Change
Adjusted P
Typed
Classe
DN496417
BU893135
CV241725
CF120135
XP_002327686
At3g46060
At5g59840
At1g53050
At4g39080
At2g18250
1.4
2.2
1.7
1.9
0.5
0.5
1.1
0.8
1.0
20.9
3.4E-02
1.3E-03
1.4E-02
3.6E-03
1.2E-03
RabE/Rab8 (1)
RabE/Rab8 (2)
RabA
VHA-a1
SNARE (1)
TGN
TGN
TGN
TGN
TGN/MVB
CK093245
CK092620
CF936851
CV277800
CV240653
At1g08560
At5g46860
At5g46860
At4g19640
At5g45130
1.3
4.0
1.8
1.1
1.1
0.4
2.0
0.8
0.1
0.1
1.1E-01
1.6E-04
3.6E-03
7.9E-01
5.2E-01
SNARE (2)
PEP12 (1)
PEP12 (2)
RabF (1)
RabF (3)
TGN/MVB
MVB
MVB
MVB
MVB
CX178052
At5g45130
1.5
0.6
7.2E-02
RabF (2)
MVB
BP932273
CV275482
At5g06140
At3g62290
0.7
2.0
20.5
1.0
1.8E-02
4.3E-03
SNX1
Arf1 (1)
RE
VF
CK088579
At3g62290
1.7
0.8
1.7E-03
Arf1 (2)
VF
BU824735
At1g60500
7.9
3.0
5.0E-05
Dynamin (1)
VF
CK090501
XP_002314626
At4g33650
At1g59820
1.9
1.1
0.9
0.1
8.7E-03
6.2E-01
Dynamin (2)
Ala-3 (1)
VF
VF
XP_002339708
At1g59820
1.8
0.8
5.1E-03
Ala-3 (2)
VF
BU810775
CV278073
XP_002306120
At1g17500
At1g32580
At4g39170
GTP-binding protein ara-3
GTP-binding protein ara-3
Ser/Thr kinase activity
Vacuolar proton ATPase subunit
Pantetheine-phosphate
adenylyltransferase
Syntaxin SYP111 (KNOLLE)
Syntaxin SYP22 (VAM3)
Syntaxin SYP22 (VAM3)
GTP-binding protein
Ras-related GTP-binding
protein RHA1
Ras-related GTP-binding
protein RHA1
Sorting nexin-like protein
ADP-ribosylation factor-like
protein
ADP-ribosylation factor-like
protein
Dynamin family protein,
GTP-binding
Dynamin-like protein ADL2
Chromaffin granule ATPase II
homolog
Chromaffin granule ATPase II
homolog
P-type ATPase
Plastid protein
SEC14-like protein
2.2
1.0
1.9
1.1
0.1
0.9
1.4E-03
8.9E-01
1.1E-03
Ala-3 (3)
DAG
SEC14
VF
VF
VF
a
Accession number of the corresponding sequence at www.ncbi.nlm.nih.gov/guide/ received from http://www.plexdb.org/modules/PD_probeset/
b
annotation.php?genechip=Poplar with Affymetrix Probe Set identifiers.
Arabidopsis Genome Initiative code of the nearest Arabidopsis homolog
a
c
b
d
e
according to .
Annotation according to .
Type of protein. Abbreviations are according to Zárský et al. (2009).
Class of protein.
MVB, Multivesicular body; RE, recycling endosome; VF, vesicle formation.
Cell Wall
Among the cell wall cluster, transcripts for esterases
and lyases, enzymes involved in pectin metabolism,
were found to be increased (Table III). Within the same
category, Leu-rich repeat proteins were elevated and
might have a function in plant defense mechanisms,
protein-protein recognition, and exocytosis (Kobe and
Deisenhofer, 1995). Transcript levels with genes encoding enzymes engaged in UDP-sugar metabolism
point to increased carbohydrate synthesis for cell wall
formation (Gibeaut and Carpita, 1994).
Hormones/Defense
Within the hormone cluster, six genes appeared to be
associated with auxin signaling (Table IV). This group
contained genes coding for pin-formed proteins (PIN)
that have been associated with auxin distribution, cell
division, cell expansion, and polar growth (Blilou et al.,
2005; Petrásek et al., 2006). Linked to vascular differentiation, three brassinosteroid metabolism genes were
found to be up-regulated in nectaries. Within the hormone cluster, two genes associated with ethylene, two
associated with JA, and five associated with salicylic
acid were induced. Interestingly, these phytohormones
represent key players in the response to wounding as
inflicted by herbivores and pathogens (Li et al., 2001;
Kachroo and Kachroo, 2007; Turner, 2007). In this
context, it should be noted that 84 of the 500 most differentially expressed genes are related to biotic stress
(Supplemental Fig. S8).
Sugar Metabolism and Transport
Nectar production demands high amounts of sugars, very likely provided by the phloem (Table V).
Because Ptt nectar-secreting cells are apoplastically
separated from the parenchyma and phloem cells (Fig.
2), sugars might be processed and supplied by the
subjacent cells. Thus, besides sugar metabolic enzymes, sugar transporters are required for unloading
from the source cells and loading into the Ptt nectarsecreting cells. Therefore, the full microarray data set
was analyzed for transcripts involved in sugar metabolism and transport. In Ptt nectaries, a cell wall and
a neutral invertase were up-regulated 10- and 6-fold,
respectively. Among the sugar transporters expressed
in Ptt nectaries, 16 hexose transporters appeared to be
Plant Physiol. Vol. 159, 2012
1183
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
Table II. qPCR monitoring of exocytosis-associated transcripts in Ptr
nectaries
nectary types is rich in tastes that are known to attract
different kinds of visitors, including bodyguards.
Comparison with microarray data from Ptt (Table I).
Typea
Classb
Fold Change,
Microarray Data
for Ptt
Fold Change,
qPCR Data for Ptr
RabE/Rab8 (1)
RabE/Rab8 (2)
RabA
VHA-a1
SNARE (1)
SNARE (2)
PEP12 (1)
PEP12 (2)
RabF (1)
RabF (2)
RabF (3)
SNX1
Arf1 (1)
Arf1 (2)
Dynamin (1)
Dynamin (2)
Ala-3 (1)
Ala-3 (2)
Ala-3 (3)
DAG
SEC14
TGN
TGN
TGN
TGN
TGN/MVB
TGN/MVB
MVB
MVB
MVB
MVB
MVB
RE
VF
VF
VF
VF
VF
VF
VF
VF
VF
1.4
2.2
1.7
1.9
0.5
1.3
4.0
1.8
1.1
1.5
1.1
0.7
2.0
1.7
7.9
1.9
1.1
1.8
2.2
1.0
1.9
0.3
0.6
0.5
0.9
1.4
0.2
0.2
0.7
0.6
0.9
0.7
0.6
0.5
0.5
0.6
0.7
0.3
0.4
0.3
0.6
1.2
a
Type of protein. Abbreviations are according to Zárský et al.
b
(2009).
Class of protein. MVB, Multivesicular body; RE, recycling
endosome; VF, vesicle formation.
induced (2- to 4-fold), but only two Suc transporters of
the suc3 type were present and not induced. In addition, a Suc synthase (SUS3) was induced by a factor of 5.
Furthermore, the analysis of the released nectar blend
revealed almost equal amounts of Glc, Fru, and Suc
(Supplemental Fig. S5). From these data, one might
speculate that (1) Suc released from the phloem is
mainly cleaved by invertases within the extracellular
space, and (2) the secretory cells predominantly import
hexose via the monosaccharide transporters, of which a
part is reconverted to Suc by SUS3. Interestingly, further sugar metabolism-related enzymes like raffinose
synthase, myoinositol oxygenase, and a-glucosidase,
which play an important role in cell wall formation and
thus reflect high cell division activity in the nectaries of
Ptt, were highly up-regulated.
DISCUSSION
Here, we provide evidence that two tree species of
the same genera might have evolved different mechanisms to control herbivory. Ptt and Ptr secrete EFN
from young leaves. Ptt nectaries operate long-term
merocrine nectar release. In contrast, Ptr grows new
nectaries for each holocrine nectar secretion event.
Thereby, the production of new nectaries on demand
in Ptr is associated with short-term but massive holocrine nectar release. The nectar composition in both
Nectary Morphology and Function
Extrafloral nectary, development, morphology, and
nectar secretion represent highly correlated items. It
has been demonstrated that the gland morphology
type determines the dose and velocity of nectar flow.
The kinetics and nectar flavor furthermore depend on
the vascular supply of basic nectar compositions, very
likely specified by factors produced as defense in the
secretory cell on demand (Díaz-Castelazo et al., 2005).
Ptr nectaries are characterized by cells covered by a
remarkably thick wall that probably impeded the free
flow of nectar. Numerous plasmodesmata between
cells in the complex likely provide for symplastic
transfer of nectar between neighboring cells. Secretory
vesicles were not observed in this nectary type. Instead, large numbers of mitochondria were found. This
feature may point to the active transport of nectar
across the plasma membrane. Microchannels in the
nectary cuticle represent a potential low-resistance
pathway for nectar secretion and were previously described in floral nectaries of Platanthera chlorantha
(Orchidaceae), Abutilon sp., and Helleborus foetidus
(Ranunculaceae; Kronestedt et al., 1986; Stpiczy
nska
et al., 2004; Koteeva, 2005). Nectaries from Ptr, after the
release of large volumes of nectar, eventually died. The
facts that (1) short-term secretion with Ptr nectaries
results in cell death and this organ type (2) does not
express granulocrine secretion-related transcripts point
to a self-destructing holocrine secretion. This mode of
secretion may thus be similar to floral nectaries of
H. foetidus or Digitalis purpurea (Gaffal et al., 2007;
Vesprini et al., 2008).
Nectaries from Ptt, in contrast, developed just one
layer of brush border-like large secretory cells (Fig. 2).
Epithelia-like nectary cells like these have not yet been
discovered in any other plant nectary type. The absence of symplastic connections between this outer cell
layer and the nectary parenchyma, in order to secrete
nectar, requires an apoplastic loading via parenchyma
cells located inside the nectary. Metabolic continuity
between the secretory cells is facilitated by a large
number of plasmodesmata in their periclinal cell walls.
The abundance of numerous secretory vesicles within
and outside of the secretory cells and the up-regulated
Ptt nectary lipid metabolism gene clusters (Fig. 8)
point to a granulocrine nectary type. In this context,
one should mention that transcriptome analysis of
Arabidopsis (Arabidopsis thaliana) floral nectaries also
suggested a granulocrine secretion mechanism (Kram
and Carter, 2009; Kram et al., 2009). Nectar production
is an expensive investment for the plant. In order to
save energy, floral nectaries of some plants are able to
reabsorb unconsumed nectar (Nepi and Stpiczy
nska,
2008). A similar situation exists in Ptt extrafloral nectaries. The lack of a cuticle and the distribution of
membrane FM dye would support nectar reabsorption
1184
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
Table III. Differentially expressed cell wall metabolism-associated genes in Ptt nectaries
Public IDa
Arabidopsis Genome
Initiative Codeb
Annotationc
Fold Change
Log2 of Fold Change
Adjusted P
BP929963
CV268088
CV281908
CV230945
CX176311
CN519614
DN502989
CV281908
CF227942
BU893810
BU811740
DN486441
CX659087
CN520892
AI165969
XP_002324379
CA927911
XP_002326320
CV244899
CV227643
CV230306
XP_002314796
At5g25610
At3g43270
At3g19320
At3g61490
At3g61490
At3g23730
At3g19320
At3g19320
At4g24780
At1g69530
At3g14310
At3g14310
At3g08900
At3g23730
At4g24780
At4g31590
At3g14310
At2g26440
At1g08200
At5g15490
At2g27860
At1g23200
RD22 (responsive to desiccation22)
Pectinesterase family protein
Leu-rich repeat family protein
Polygalacturonase (pectinase) family protein
Polygalacturonase (pectinase) family protein
Endo-xyloglucan transferase, putative
Leu-rich repeat family protein
Leu-rich repeat family protein
Pectate lyase family protein
ATEXPA1 (Arabidopsis expansin A1)
ATPME3 (Arabidopsis pectin methylesterase3)
ATPME3 (Arabidopsis pectin methylesterase3)
RGP3 (reversibly glycosylated polypeptide3)
Endo-xyloglucan transferase, putative
Pectate lyase family protein
ATCSLC05 (cellulose synthase-like C5)
ATPME3 (Arabidopsis pectin methylesterase3)
Pectinesterase family protein
AXS2 (UDP-D-apiose/UDP-D-Xyl synthase2)
UDP-Glc-6-dehydrogenase, putative
AXS1 (UDP-D-apiose/UDP-D-Xyl synthase1)
Pectinesterase family protein
22.9
15.2
10.0
10.0
8.7
8.5
8.0
7.5
6.6
6.3
5.3
4.9
4.9
4.9
4.2
4.2
4.0
3.9
3.8
3.6
3.5
0.1
4.5
3.9
3.3
3.3
3.1
3.1
3.0
2.9
2.7
2.7
2.4
2.3
2.3
2.3
2.1
2.1
2.0
2.0
1.9
1.9
1.8
23.0
5.4E-05
2.6E-05
6.6E-05
4.4E-05
5.5E-05
2.6E-05
7.1E-05
7.8E-05
2.9E-05
3.7E-05
8.2E-05
3.3E-05
5.8E-05
8.2E-05
5.1E-05
5.4E-05
8.2E-05
8.2E-05
5.8E-05
8.3E-05
8.2E-05
2.6E-05
a
Accession number of the corresponding sequence at www.ncbi.nlm.nih.gov/guide/ received from http://www.plexdb.org/modules/PD_probeset/
b
annotation.php?genechip=Poplar with Affymetrix Probe Set identifiers.
Arabidopsis Genome Initiative code of the nearest Arabidopsis homolog
c
according to a.
Annotation according to b.
via endocytosis of cells in the outer layer of the secretory organ. Here, bulk secretion seems to be mediated by multivesicular bodies (Fig. 2), components of
the plant exocytosis system (Foresti et al., 2008; Foresti
and Denecke, 2008; Robinson et al., 2008; Zárský et al.,
2009). Accordingly microarray studies with Ptt nectaries
identified an enrichment of transcripts involved in
granulocrine secretion. In Ptr nectaries, however, the
same genes appeared not to be induced. Moreover, Ptr
nectaries run dry after a few days. Another nectar-release
cycle, therefore, depends on the production of a new set
of holocrine-secreting nectaries. Our findings with the
Table IV. Differentially expressed hormone metabolism-associated genes in Ptt nectaries
Public IDa
Arabidopsis Genome
Initiative Codeb
Annotationc
Fold Change
Log2 of Fold
Change
Adjusted P
hord
XP_002312924
XP_002316954
XP_002319398
XP_002306218
CV237921
XP_002336658
CK092098
CA823181
AJ778035
XP_002300561
CX184416
DN490862
BP933461
CV262113
BU825949
CV263317
XP_002335942
XP_002334262
CA929119
At1g29430
At5g54510
At5g54510
At1g29510
At1g77690
At5g16530
At1g75080
At3g19820
At3g19820
At4g20880
At2g40940
At1g06620
At5g42650
At1g55020
At4g36470
At4g36470
At5g38020
At1g19640
At1g68040
Auxin-responsive family protein
DFL1/GH3.6 (dwarf in light1)
DFL1/GH3.6 (dwarf in light1)
SAUR68 (small auxin up-regulated68)
Amino acid permease, putative
PIN5 (pin-formed5); auxin:hydrogen symporter
BZR1 (brassinazole-resistant1)
DWF1 (diminuto1); catalytic
DWF1 (diminuto1); catalytic
Ethylene-regulated nuclear protein (ERT2)
ERS1 (ethylene response sensor1); receptor
2-Oxoglutarate-dependent dioxygenase, putative
AOS (allene oxide synthase)
LOX1 (lipoxygenase1)
BSMT1; S-adenosyl-Met-dependent methyltransferase
BSMT1; S-adenosyl-Met-dependent methyltransferase
BSMT1; S-adenosyl-Met-dependent methyltransferase
JMT (jasmonic acid carboxyl methyltransferase)
Carboxyl methyltransferase family protein
3.4
15.7
10.7
5.2
5.1
6.7
4.2
6.3
4.8
5.2
4.1
0.1
5.0
3.5
40.0
30.7
22.7
25.2
15.9
1.8
4.0
3.4
2.4
2.3
2.8
2.1
2.7
2.3
2.4
2.0
23.5
2.3
1.8
5.3
4.9
4.5
4.7
4.0
6.5E-05
3.3E-05
4.9E-05
3.9E-05
8.2E-05
4.4E-05
8.4E-05
5.5E-05
5.6E-05
7.4E-05
6.4E-05
2.6E-05
8.2E-05
7.4E-05
2.3E-05
3.4E-05
4.3E-05
6.9E-05
2.6E-05
aux
aux
aux
aux
aux
aux
bra
bra
bra
eth
eth
eth
JA
JA
SA
SA
SA
SA
SA
a
Accession number of the corresponding sequence at www.ncbi.nlm.nih.gov/guide/ received from http://www.plexdb.org/modules/PD_probeset/
b
Arabidopsis Genome Initiative code of the nearest Arabidopsis homolog
annotation.php?genechip=Poplar with Affymetrix Probe Set identifiers.
c
d
according to a.
Annotation according to b.
Related to the hormone auxin (aux), brassinosteroids (bra), ethylene (eth), JA, or salicylic acid (SA).
Plant Physiol. Vol. 159, 2012
1185
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
Table V. Sugar metabolism- and transport-associated genes in Ptt nectaries
Public IDa
Major carbohydrate metabolism
BU879414
CV260807
BP928698
Minor carbohydrate metabolism
BP933945
XP_002330589
CV271186
Sugar transport
CV267500
CV265114
CA823118
Arabidopsis
Genome
Initiative Codeb
Annotationc
Fold Change Log2 of Fold Change Adjusted P
At3g52600
At3g05820
At4g02280
ATCWINV2, CWIN4, cell wall invertase
Alkaline/neutral invertase H
ATSUS3, Suc synthase3
10.1
6.2
4.8
3.3
2.6
2.3
2.8E-04
8.2E-05
3.7E-05
At2g19800
At1g55740
At5g08370
Myoinositol oxygenase2
Raffinose synthase1
a-Galactosidase2
75.5
21.0
4.2
6.2
4.4
2.1
2.8E-05
4.1E-05
6.4E-05
At5g16150
At2g43330
At1g73220
GLT1, Glc transporter1
ATINT1, inositol transporter1
ATOCT1, organic cation/carnitine
transporter1
ATPLT5, polyol/monosaccharide
transporter5
Sugar transporter family protein
STP13, sugar transport protein13
Putative sugar/inositol transport protein
ATINT4, inositol transporter4
Sugar transporter family protein
TMT2, tonoplast monosaccharide
transporter2
Nucleotide-sugar transporter family protein
ATUTR6, UDP-Gal transporter6
STP13, sugar transport protein13
Sugar transporter family protein
ATOCT1, organic cation/carnitine
transporter1
Hexose transporter, putative
ATSUC3, Suc transporter3
ATSUC3, Suc transporter3
3.9
3.4
2.8
2.0
1.8
1.5
1.7E-04
1.5E-04
2.0E-04
2.7
1.4
4.3E-04
2.5
2.5
2.4
2.3
2.2
2.2
1.3
1.3
1.2
1.2
1.1
1.1
2.2E-03
2.6E-03
2.8E-04
2.6E-03
1.6E-03
7.4E-03
2.2
2.1
2.1
2.0
2.0
1.1
1.1
1.1
1.0
1.0
9.3E-04
2.4E-03
8.9E-04
2.2E-03
2.8E-03
2.0
1.5
1.5
1.0
0.6
0.6
4.6E-03
4.6E-03
9.7E-03
XP_002313809
At3g18830
CV237054
CB307011
XP_002310808
XP_002308798
CK107634
CV241535
At5g17010
At5g26340
At1g75220
At4g16480
At5g17010
At4g35300
CV272388
CA928662
CV279380
BU866894
XP_002329696
At5g41760
At3g59360
At5g26340
At5g59250
At1g73220
XP_002324427
XP_002315798
XP_002311596
At1g67300
At2g02860
At2g02860
a
Accession number of the corresponding sequence at www.ncbi.nlm.nih.gov/guide/ received from http://www.plexdb.org/modules/PD_probeset/
b
Arabidopsis Genome Initiative code of the nearest Arabidopsis homolog
annotation.php?genechip=Poplar with Affymetrix Probe Set identifiers.
a
c
b
according to .
Annotation according to .
tree model Populus thus seem to support the notion that
Ptt and Ptr operate two different nectary types and secretion mechanisms.
Appearance of Nectaries and Nectar
In wild cotton (Gossypium thurberi), the size and
density of extrafloral nectaries are heritable (Rudgers
and Strauss, 2004). One report focusing on P. tremuloides ecotypes with extrafloral nectary induction has
suggested a genetic component (Wooley et al., 2007).
This study showed that nectaries were generally more
abundant on younger than on older leaves and decreased with tree age. Our finding that 4-year-old Ptt
trees expressed nectaries on about 40% of the leaves is
well in agreement with data that were found with 4- to
5-year-old trees (Wooley et al., 2007). However, we
cannot exclude that the number of Ptt nectaries might
increase under massive herbivore attack, as predicted
by Wooley et al. (2007). In spring, nectaries appeared
on most emerging leaves of both species, and they
seem to protect Ptt as well as Ptr leaves from herbivore
attack (Fig. 3; Supplemental Fig. S4). Secondary nectar
release from Ptr was observed in response to the invasion of phloem-feeding insects. Poplar species are
known to have several direct defense mechanisms
against herbivores, like the production of condensed
tannins, phenolic glycosides, and salicortin (Hwang
and Lindroth, 1998; Osier et al., 2000; Donaldson and
Lindroth, 2007). Direct and indirect defenses are costly.
The inducible expression of secondary nectaries with
Ptr might thus be a strategy to avoid interference of the
different defense systems. Recent studies indicate that
continuous mechanical tissue damage is sufficient to
trigger JA production and subsequent volatile emission (Mithöfer et al., 2005). In addition to wounding,
chemical elicitors present in insect saliva also may play
an important role in the extrafloral nectary response
(Radhika et al., 2010). Upon wounding of Ptr, a typical
JA or herbivory-linked VOC spectrum was emitted,
but new nectaries or the release of nectar from existing
nectaries were not observed. These findings clearly
indicate that neither jasmonates nor induced volatiles
1186
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
trigger Ptr nectar production, as was reported previously
in lima beans (Heil et al., 2008). Herbivore-induced Ptr
VOCs, however, may be used directly by carnivorous
arthropods as a cue for host localization or as a plantplant signaling mechanism, as proposed (Heil et al.,
2008). This also implies that the mealy bug-induced
nectar production and nectary formation is linked to
currently unknown signaling pathways.
Nectar Blend
Gross chemical properties of nectars tend to be
similar in plants attracting the same animals. The EFN
of myrmecophyte Acacia contains just Glc and Fru. It
has been shown that the addition of Suc significantly
changed the attractiveness of this nectar (Heil et al.,
2005). Cell wall invertases (CWIN) are important determinants of sink strength and, thus, phloem sugar
unloading and the suppression of reloading (Roitsch,
1999). Recently, CWIN4 was described as the first cell
wall invertase that is required for nectar production in
Arabidopsis (Kram et al., 2009; Ruhlmann et al., 2010).
In the nectary transcriptome of this model plant, CWIN4associated sugar synthases appeared increased. Similarly, Ptt nectaries up-regulate genes encoding invertases,
hexose transporters, and sugar synthases. In addition,
an invertase sharing high homologies with CWIN2
as well as CWIN4 was found 10-fold up-regulated. In
agreement with the aforementioned Arabidopsis
studies, our data give rise to the hypothesis that with
Ptt nectaries, sugar is mainly imported and further
processed by secretory cells as monosaccharide. The
fact that Ptt nectar contains equal amounts of Fru, Glc,
and Suc strengthens this theory. Nectar of Ptt might
thus be considered as tasty for generalists, which appeared to be reflected by the diversity of visitors described for P. tremula (Wooley et al., 2007). Ptr, in
contrast, seems to be suited for specialized visitors.
Thus, insights into the kind of visitors might already
be gained by the nectar blend. Analyzing the nature of
the visitors as a function of nectary type, nectar composition, and bodyguards represents a future goal.
Thereby, the question will be addressed whether
poplar plants can shape the blend of the nectar in an
insect-dependent manner.
In this respect, amino acids represent tasty nectar
components (Baker and Baker, 1973, 1983). The EFNs
from Ptt and Ptr were rich in “sweet” taste amino acids
like Phe, which is the most abundant amino acid in
nectars of bee-pollinated plants and also is known to
serve as a bee phagostimulant (Inouye and Waller,
1984; Petanidou, 2007). Pro has the unique ability to
stimulate the insect’s salt cell, which results in enhanced feeding behavior (Hansen et al., 1998; Wacht
et al., 2000). We found Pro in EFN of both poplar
species but not in leaf extracts. It is by far the most
abundant amino acid in honeybee hemolymphe, important for egg laying (Hrassnigg et al., 2003), and
regulates the conversion of nectar into honey (Davies,
1978). Therefore, the composition of Ptt nectar seemed
suitable to support the honeybee lifestyle. It has been
shown that honeybees, parasitic flies (Tachinidae),
parasitic wasps (Ichneumonidae), and bees are common visitors of all poplar nectaries (Supplemental Fig.
S6; Trelease, 1881; Wooley et al., 2007). Flying honeybees and wasps produce similar air disturbances that
stimulate sensory hairs of many caterpillars. As a result, caterpillars stop moving or drop off the plant.
Thereby, the feeding intensity of the herbivores is reduced (Tautz, 1977; Tautz and Markl, 1978; Tautz and
Rostas, 2008). Attracting honeybees and wasps with
tasty nectar, therefore, might be an effective strategy to
reduce poplar leaf damage by herbivore infestation.
CONCLUSION
The morphology of Ptt nectaries revealed granulocrine secretion via multivesicular bodies, whereas Ptr
showed typical multilayer secretory cells with a structure similar to floral nectaries. Ptt continuously secretes
nectar from long-living nectaries, and excess nectar is
reabsorbed via endocytosis. Thus, membrane trafficking is frequent and might explain why only one distinct
layer of secretory cells is present in these nectaries. Both
Ptt and Ptr trees seem to protect their delicate first
leaves in spring against herbivores by nectar production. In contrast to continuously nectar-secreting Ptt,
Ptr, only in the case of special insect attack, produces
secondary nectar by extrafloral nectaries. The emerging
Ptr nectar seems to attract rather specialist visitors,
maybe depending on the particular herbivore. Ptt, in
contrast, provides nectar for generalists according to
unspecific nectar production.
These different defense strategies require varying
secretion systems, which we confirmed by transcript
and metabolite compositions, as well as the morphology and physiology of nectaries and nectar of both
poplar species.
MATERIALS AND METHODS
Plant Material and Growing Conditions
Populus tremula 3 Populus tremuloides plants (clone T89) and Populus trichocarpa (clone 93-968) were field grown in soil under natural conditions in the
Botanical Garden of Wuerzburg or cultivated under long-day conditions in
climate chambers (16 h of light [22°C]/8 h of darkness [17°C]; TLD 58 W/840
Super 80 [Philips] and 58 W L58/77 [Osram] lamps). Field-grown trees used
for effectiveness tests and the visitor determinations were about 3 to 4 years
old and 15 to 20 feet high. All other experiments were performed using cultivated trees of about 4 to 5 feet in height. These plants were watered twice a
week and fertilized frequently.
Light Microscopy
Nectaries from Ptr or Ptt were harvested and fixed by passing through
ascending grades of ethanol for 45 min each. After dehydration, nectaries were
embedded in 2-hydroxyethyl methacrylate/glycol methacrylate (Agar Scientific) according to the manufacturer’s advice. Sections of extrafloral nectaries
(20 mm) were cut with a c-profile 16-cm knife (Leica) in a Leica RM2165 microtome and heat fixed to microscope slides. Specimens were stained with
Plant Physiol. Vol. 159, 2012
1187
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
toluidine blue, mounted in immersion oil under coverslips, and examined
with a VHX-100k digital microscope (Keyence Corp.).
Nectary Appearance and Effectiveness
Nectary appearance and leaf damage were quantified using 11 randomly
chosen branches (1,257 leaves in total) of three different trees (Ptt) or six
branches (327 leaves in total) of two different trees. Damage was classified as
severe (more than 75% damage of the leaf surface), mild (less than 75%), or
healthy (no damage).
TEM
Small sections of leaf tissue (1–2 mm) were cut with a razor blade and
immediately immersed for 4 h in fixation medium containing 1% (w/v) formaldehyde, 1 mM EGTA, 50 mM cacodylate buffer, and 5% glutaraldehyde.
Subsequently, the tissue was postfixed with 2% (w/v) osmium tetroxide
overnight at room temperature, stained with 3% (w/v) uranyl acetate in 20%
ethanol for 1 h, dehydrated in a graded series of ethanol, and embedded in
Spurr’s epoxy resin (Spurr, 1969). Ultrathin sections with a thickness of 70 to
80 nm were cut with a diamond knife on an ultramicrotome (Ultratome Nova;
LKB), transferred onto formvar-coated copper grids, and stained for 10 min
with lead citrate. Sections were examined using a Zeiss EM 10c transmission
electron microscope at 80 kV.
Quantification of Nectar Sugars and Amino Acids
For nectar sugar quantification, different amounts of nectar were diluted in
HPLC water (Sigma) to a final volume of 1 mL, boiled for 5 min, and
centrifuged (10 min at 14,000g). The supernatant was treated with Serdolit
MB1 (Serva; 10 mg 100 mL21 sample), and the sugar concentration was measured using a pulse electrochemical detector (Dionex 4500i). Amino acid
quantity and quality were measured using an amino acid analyzer (Biochrom
20 Plus).
Induction of EFN
The experiment was conducted under natural conditions with about 1-yearold soil-grown Ptr. The four youngest fully expanded leaves of each plant
were induced, either by puncturing 100 times with a needle (1 mm diameter)
or by cutting the leaf tip (about 10% of the total leaf area). Plants were observed using a Keyence VHX-100k digital microscope. Every 15 min, photographs were taken. For “herbivory induction,” caterpillars (Spodoptera littoralis,
Spodoptera exigua, and Lymantria dispar) were placed on Ptr leaves.
Induction of Volatile Biosynthesis with JA or Coronalon
Leaves of the test plants were sprayed with aqueous solutions of JA (1 and
0.5 mM) or coronalon (0.1 mM) until runoff. After drying (1 h), the pretreated
plants were enclosed in PET foil bags, and volatile collection was achieved
over 24 h as described. If the volatile collection was extended to 48 h, the plant
leaves were sprayed and dried a second time after 24 h.
Volatile Induction by Herbivorous Insects
Larvae of S. littoralis (Lepidoptera, Noctuidae) were used as herbivores for
volatile induction. For feeding experiments, third-instar larvae were used. Five
larvae were placed on a plant, and the test plant was enclosed in a PET foil
bag. Volatiles were then collected as described.
Continuous Mechanical Damage of Plant Leaves
using MecWorm
Individual leaves of the intact test plant were continuously damaged by the
robotic MecWorm system (Mithöfer et al., 2005) over a period of 24 h, resulting
in 333 mm2 of damaged area using four punches per minute. Volatiles were
collected as described. Additional experiments were conducted by combining
continuous mechanical damage with the addition of oral secretions from the
larvae (1:10 dilution) to the damaged area.
RNA Isolation and Amplification for Microarrays
Total RNA of leaves was extracted using the E.Z.N.A. Plant RNA Mini Kit
(V Bio-Tek) and nectary total RNA with the RNeasy MicroKit (Qiagen),
according to the manufacturers’ protocols, with minor modifications. The
binding buffers were supplemented with 2% b-mercaptoethanol, 1% polyvinylpyrrolidone, and 70 mM potassium ethylxantogenate and added to 30 mg
of homogenized leaf tissue or 120 homogenized nectaries, respectively. After
incubation at room temperature for 30 min, the sample were briefly centrifuged before transfer to the individual homogenation columns. One microgram of total RNA was used for RNA amplification based on the BD-SMART
mRNA amplification kit (BD Bioscience). The mRNA was reverse transcribed
using SuperScript II Reverse Transcriptase (Invitrogen). To preamplify fulllength complementary DNAs (cDNAs) prior to in vitro transcription, an additional 10-cycle PCR (95°C for 30 s, 60°C for 1 min, and 68°C for 10 min) was
introduced using t7 extension and PCR primer IIA of the kit. RNA integrity
and concentration were monitored using the Experion Automated Electrophoresis System (Bio-Rad) with the Experion RNA Highsense Analysis Kit
according to the manufacturer’s protocol.
qPCR
Collection and Analysis of Volatiles
The collection of volatiles emitted from leaves of the test plants damaged by
caterpillars or MecWorm or treated with chemical elicitors (JA or coronalon)
was achieved using charcoal traps. Elicitor-treated or insect-damaged areas of
the plants were enclosed in polyethylene terephthalate (PET) foil bags that
were tightly closed at both ends to guarantee the accumulation of volatiles in
the enclosed volume and to avoid contamination with volatiles from the
potting soil. One end of the PET bags was connected for 24 h to a volatile
collection device (Kunert et al., 2009), and the emitted volatiles were trapped
on charcoal filters (1.5 mg; CLSA Filter; Gränicher & Quartero) by air circulation maintained by a circulation pump. The trapped volatiles (24, 48, or 72 h)
were desorbed from the filters using 2 3 20 mL of dichloromethane containing
1-bromodecane (50 mg mL21) as an internal standard. An aliquot (1 mL) of the
stock solution was analyzed on a Finnigan Trace gas chromatography-mass
spectrometry device equipped with an EC-5 column (15 m 3 0.25 mm 3 0.25
mm; Alltech). Helium at a flow rate of 1.5 mL min21 served as a carrier gas.
The gas chromatography injector, transfer line, and ion source were set at
220°C, 280°C, and 280°C, respectively. Volatiles were separated under programmed conditions using a temperature profile from 40°C (2 min) at 10°C
min21 to 200°C and at 30°C min21 to 280°C. The split ratio of the stock solution
was 1:10, and the resulting split flow was 15 mL min21 (10 times the column
gas flow of 1.5 mL min21). Authentic standards were used for the identification of compounds.
To remove any remaining DNA, total RNA was treated with RNase-free
DNase (Fermentas) according to the manufacturer’s protocol. First-strand
cDNA was prepared using 2.5 mg of RNA with the Moloney murine leukemia virus reverse transcriptase kit (Promega). First-strand cDNA was
20-fold diluted for reverse transcription-PCR. qPCR was performed in a
Mastercycler ep Realplex2 S (Eppendorf) with the ABsolute QPCR SYBR
Green Capillary Mix (ABgene) in a 20-mL reaction volume. After a “hot
start” (15 min at 95°C), a standard PCR program was applied: 40 times
for 15 s at 95°C, 15 s at primer-specific annealing temperature, and 20 s at
72°C, followed by a dissociation curve (10 s at 95°C, followed by a temperature ramp from 60°C–95°C with an increment of 0.3°C s 21 ). Primers
used (TIB MOLBIOL) have been designed for Ptt or Ptr and were tested
regarding optimal annealing temperature, specificity by dissociation
curves, and gel electrophoresis (data not shown) prior to qPCR. Primers
are listed in Supplemental Table S3. All quantifications were normalized
to actin cDNA fragments amplified by PtACT2fwd and PtACT2rev. These
fragments are homologous to the constitutively expressed Arabidopsis
(Arabidopsis thaliana) Actin2 and Actin8 (for details, see An et al., 1996;
Szyroki et al., 2001). Each transcript was quantified using individual
standards. To enable the detection of contaminating genomic DNA, PCR
was performed with the same RNA as template, which was used for
cDNA synthesis. All kits were used according to the manufacturer’s
protocols.
1188
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
Microarrays
LITERATURE CITED
Microarray analyses were conducted at the Microarray Facility, University
of Tübingen. Samples of leaves and nectaries from Ptt field culture were analyzed. All samples were amplified using the One-Cycle Target Labeling
Assay (Affymetrix) according to the manufacturer’s protocol and hybridized
to the Gene Chip Poplar Genome (Affymetrix). Microarrays were scanned
using the GCS3000 GeneChip scanner (Affymetrix) and GCOS software version 1.4. Scanned images were subjected to visual inspection to control for
hybridization artifacts and proper grind alignment. Files for quality control
were generated using the program Expression Console (Affymetrix).
An YQ, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher
RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8
actin subclass in vegetative tissues. Plant J 10: 107–121
Arimura G, Huber DP, Bohlmann J (2004) Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid
volatiles in hybrid poplar (Populus trichocarpa 3 deltoides): cDNA
cloning, functional characterization, and patterns of gene expression of
(2)-germacrene D synthase, PtdTPS1. Plant J 37: 603–616
Arimura G, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J
(2000) Herbivory-induced volatiles elicit defence genes in lima bean
leaves. Nature 406: 512–515
Baker HG, Baker I (1973) Amino-acids in nectar and their evolutionary
significance. Nature 241: 543–545
Baker HG, Baker I (1983) A brief historical review of the chemistry of floral
nectar. In B Bentley, T Elias, eds, The Biology of Nectaries. Columbia
University Press, New York, pp 126–152
Bashkirov PV, Akimov SA, Evseev AI, Schmid SL, Zimmerberg J, Frolov
VA (2008) GTPase cycle of dynamin is coupled to membrane squeeze
and release, leading to spontaneous fission. Cell 135: 1276–1286
Benjamini Y, Hochberg Y (2000) On the adaptive control of the false discovery rate in multiple testing with independent statistics. J Educ Behav
Stat 25: 60–83
Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R,
Aida M, Palme K, Scheres B (2005) The PIN auxin efflux facilitator
network controls growth and patterning in Arabidopsis roots. Nature
433: 39–44
Boland W, Hopke J, Donath J, Nuske J, Bublitz F (1995) Jasmonic acid and
coronatin induce odor production in plants. Angew Chem Int Ed Engl
34: 1600–1602
Brandenburg A, Dell’Olivo A, Bshary R, Kuhlemeier C (2009) The
sweetest thing: advances in nectar research. Curr Opin Plant Biol 12:
486–490
Chamberlain SA, Holland JN (2009) Quantitative synthesis of context
dependency in ant-plant protection mutualisms. Ecology 90: 2384–2392
D’Amato F (1984) Role of polyploidy in reproductive organs and tissues. In
BM Johri, ed, Embryology of Angiosperms. Springer, Berlin, pp 519–566
Danner H, Boeckler GA, Irmisch S, Yuan JS, Chen F, Gershenzon J,
Unsicker SB, Köllner TG (2011) Four terpene synthases produce major
compounds of the gypsy moth feeding-induced volatile blend of Populus
trichocarpa. Phytochemistry 72: 897–908
Dauwalder M, Whaley WG (1982) Membrane assembly and secretion in
higher plants. J Ultrastruct Res 78: 302–320
Davies AMC (1978) Proline in honey: osmoregulatory hypothesis. J Apic
Res 17: 227–233
De Moraes CM, Mescher MC, Tumlinson JH (2001) Caterpillar-induced
nocturnal plant volatiles repel conspecific females. Nature 410: 577–580
Díaz-Castelazo C, Rico-Gray V, Ortega F, Angeles G (2005) Morphological
and secretory characterization of extrafloral nectaries in plants of coastal
Veracruz, Mexico. Ann Bot (Lond) 96: 1175–1189
Donaldson JR, Lindroth RL (2007) Genetics, environment, and their interaction determine efficacy of chemical defense in trembling aspen.
Ecology 88: 729–739
Escalante-Pérez M, Heil M (2012) Nectar secretion: its ecological context
and physiological regulation. In J.M. Vivanco, F Baluska, eds, Secretions
and Exudates in Biological Systems. Springer, Berlin, pp 187–220
Fahn A (1988a) Secretory tissues and factors influencing their development.
Phyton Ann Rei Bot 28: 13–26
Fahn A (1988b) Secretory tissues in vascular plants. New Phytol 108: 229–257
Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA,
Read ND (2000) Confocal microscopy of FM4-64 as a tool for analysing
endocytosis and vesicle trafficking in living fungal hyphae. J Microsc
198: 246–259
Foresti O, De Marchis F, de Virgilio M, Klein EM, Arcioni S, Bellucci M,
Vitale A (2008) Protein domains involved in assembly in the endoplasmic reticulum promote vacuolar delivery when fused to secretory
GFP, indicating a protein quality control pathway for degradation in the
plant vacuole. Mol Plant 1: 1067–1076
Foresti O, Denecke J (2008) Intermediate organelles of the plant secretory
pathway: identity and function. Traffic 9: 1599–1612
Biostatistical Analyses
Data preprocessing was performed using the Bioconductor software
(Gentleman et al., 2004) with the statistical programming environment R
(Ihaka and Gentleman, 1996). Background correction and normalization were
performed using the variance stabilization method (variance stabilization
normalization [vsn] and robust multichip average [rma]; Huber et al., 2002),
and probe-set summaries were calculated with the medianpolish algorithm of
rma (Irizarry et al., 2003). Exploratory analysis by hierarchical clustering of the
arrays and correspondence analysis suggested the removal of one leaf array as
an outlier, leaving a batch of five confident arrays in total. Differential expression between nectaries and leaves has been calculated using the moderated t statistic implemented in the eBayes function of the Limma package
(Smyth, 2004), which was specifically developed for the analysis of smallsample-size experiments. By exploiting information across genes, it delivers
more stable results than a conventional t test. The P values of all results were
corrected for multiple testing by applying the false discovery rate from
Benjamini and Hochberg (2000).
The 500 most differentially expressed genes were analyzed by MapMan
3.1.1. The Affymetrix probe set identifiers were imported and mapped with
Populus trichocarpa/Ptrich_AFFY_09:1.0 and the pathway overview 1.0
(http://mapman.gabipd.org/web/guest). Annotation of Affymetrix probe set
identifiers was done by using BarleyBase/PLEXdb (Wise et al., 2007).
Microarray data have been uploaded to the Gene Expression Omnibus. Data
are accessible at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=pzehnqawcmikqxw&acc=GSE23897.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Enlarged TEM images of Ptt and Ptr nectaries.
Supplemental Figure S2. Ptt nectaries stained with Sudan III.
Supplemental Figure S3. Fluorescence microscopy of Ptt nectary longitudinal sections stained with FM4-64.
Supplemental Figure S4. Ptr nectary density and effectiveness against herbivore attack.
Supplemental Figure S5. Sugar content in nectars of Ptt and Ptr.
Supplemental Figure S6. Visitors attracted by extrafloral Ptt and Ptr nectaries.
Supplemental Figure S7. The most abundant amino acids in leaves of Ptt
and Ptr.
Supplemental Figure S8. MapMan gene clusters involved in biotic stress
responses.
Supplemental Table S1. List of 500 differentially expressed genes in Ptt
nectaries versus leaves.
Supplemental Table S2. Validation of microarray data by qPCR.
Supplemental Table S3. Primers used in qPCR.
Supplemental Protocol S1. Sudan III and FM-64 staining.
ACKNOWLEDGMENTS
We thank Martin Heil for critical reading of the manuscript.
Received February 22, 2012; accepted May 8, 2012; published May 9, 2012.
Plant Physiol. Vol. 159, 2012
1189
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Escalante-Pérez et al.
Gaffal KP, Friedrichs GJ, El-Gammal S (2007) Ultrastructural evidence for
a dual function of the phloem and programmed cell death in the floral
nectary of Digitalis purpurea. Ann Bot (Lond) 99: 593–607
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S,
Ellis B, Gautier L, Ge Y, Gentry J, et al (2004) Bioconductor: open
software development for computational biology and bioinformatics.
Genome Biol 5: R80
Gershenzon J (2007) Plant volatiles carry both public and private messages.
Proc Natl Acad Sci USA 104: 5257–5258
Gibeaut DM, Carpita NC (1994) Biosynthesis of plant cell wall polysaccharides. FASEB J 8: 904–915
Hansen K, Wacht S, Seebauer H, Schnuch M (1998) New aspects of chemoreception in flies. Ann N Y Acad Sci 855: 143–147
Heil M (2008) Indirect defence via tritrophic interactions. New Phytol 178:
41–61
Heil M (2011) Nectar: generation, regulation and ecological functions.
Trends Plant Sci 16: 191–200
Heil M, Koch T, Hilpert A, Fiala B, Boland W, Linsenmair K (2001) Extrafloral nectar production of the ant-associated plant, Macaranga tanarius, is an induced, indirect, defensive response elicited by jasmonic
acid. Proc Natl Acad Sci USA 98: 1083–1088
Heil M, Lion U, Boland W (2008) Defense-inducing volatiles: in search of
the active motif. J Chem Ecol 34: 601–604
Heil M, Rattke J, Boland W (2005) Postsecretory hydrolysis of nectar sucrose and specialization in ant/plant mutualism. Science 308: 560–563
Heil M, Silva Bueno JC (2007) Within-plant signaling by volatiles leads to
induction and priming of an indirect plant defense in nature. Proc Natl
Acad Sci USA 104: 5467–5472
Hrassnigg N, Leonhard B, Crailsheim K (2003) Free amino acids in the
haemolymph of honey bee queens (Apis mellifera L.). Amino Acids 24:
205–212
Huber W, von Heydebreck A, Sültmann H, Poustka A, Vingron M (2002)
Variance stabilization applied to microarray data calibration and to the
quantification of differential expression. Bioinformatics (Suppl 1) 18:
S96–S104
Hwang SY, Lindroth RL (1998) Consequences of clonal variation in aspen
phytochemistry for late season folivores. Ecoscience 5: 508–516
Ihaka R, Gentleman R (1996) R: a language for data analysis and graphics.
J Comput Graph Statist 5: 299–314
Inouye DW, Waller GD (1984) Responses of honey bees (Apis mellifera) to
amino acid solutions mimicking floral nectars. Ecology 65: 618–625
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf
U, Speed TP (2003) Exploration, normalization, and summaries of high
density oligonucleotide array probe level data. Biostatistics 4: 249–264
Jones CE (1983) Nectar production: the biology of nectaries. Science 221:
1172
Jürgens G, Geldner N (2002) Protein secretion in plants: from the transGolgi network to the outer space. Traffic 3: 605–613
Kachroo A, Kachroo P (2007) Salicylic acid-, jasmonic acid- and ethylenemediated regulation of plant defense signaling. Genet Eng (N Y) 28: 55–83
Kaczorowski RL, Juenger TE, Holtsford TP (2008) Heritability and correlation structure of nectar and floral morphology traits in Nicotiana
alata. Evolution 62: 1738–1750
Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the
emerging molecular analysis. Annu Rev Plant Biol 53: 299–328
Kobe B, Deisenhofer J (1995) Proteins with leucine-rich repeats. Curr Opin
Struct Biol 5: 409–416
Kost C, Heil M (2008) The defensive role of volatile emission and extrafloral nectar secretion for lima bean in nature. J Chem Ecol 34: 1–13
Koteeva NK (2005) A novel structural type of plant cuticle. Dokl Biol Sci
403: 272–274
Kram BW, Carter CJ (2009) Arabidopsis thaliana as a model for functional
nectary analysis. Sex Plant Reprod 22: 235–246
Kram BW, Xu WW, Carter CJ (2009) Uncovering the Arabidopsis thaliana
nectary transcriptome: investigation of differential gene expression in
floral nectariferous tissues. BMC Plant Biol 9: 92
Kronestedt EC, Robards AW, Stark M, Olesen P (1986) Development of
trichomes in the abutilon nectary gland. Nord J Bot 6: 627–639
Kunert M, David A, Becher J, Boland W (2009) Volatile sampling from
biological sources by the closed-loop-stripping technique. Cold Spring
Harb Protoc 2009: pdb prot5233
Lawrence SD, Dervinis C, Novak N, Davis JM (2006) Wound and insect
herbivory responsive genes in poplar. Biotechnol Lett 28: 1493–1501
Li L, Li C, Howe GA (2001) Genetic analysis of wound signaling in tomato:
evidence for a dual role of jasmonic acid in defense and female fertility.
Plant Physiol 127: 1414–1417
Linsenmair KE, Heil M, Kaiser WM, Fiala B, Koch T, Boland W (2001)
Adaptations to biotic and abiotic stress: Macaranga-ant plants optimize
investment in biotic defence. J Exp Bot 52: 2057–2065
Litvak V, Dahan N, Ramachandran S, Sabanay H, Lev S (2005) Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2
protein is critical for Golgi secretory function. Nat Cell Biol 7: 225–234
Mathews CR, Brown MW, Bottrell DG (2007) Leaf extrafloral nectaries
enhance biological control of a key economic pest, Grapholita molesta
(Lepidoptera: Tortricidae), in peach (Rosales: Rosaceae). Environ Entomol 36: 383–389
Mithöfer A, Wanner G, Boland W (2005) Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile
emission. Plant Physiol 137: 1160–1168
Nepi M, Stpiczy
nska M (2007) Nectar resorption and translocation in
Cucurbita pepo L. and Platanthera chlorantha Custer (Rchb.). Plant Biol
(Stuttg) 9: 93–100
Nepi M, Stpiczy
nska M (2008) The complexity of nectar: secretion and
resorption dynamically regulate nectar features. Naturwissenschaften
95: 177–184
Osier TL, Hwang SY, Lindroth RL (2000) Effects of phytochemical variation in quaking aspen Populus tremuloides clones on gypsy moth Lymantria dispar performance in the field and laboratory. Ecol Entomol 25:
197–207
Pacini E, Nepi M, Vesprini JL (2003) Nectar biodiversity: a short review.
Plant Syst Evol 238: 7–21
Petanidou T (2007) Ecological and evolutionary aspects of floral nectars in
Mediterranean habitats. In S Nicolson, M Nepi, E Pacini, eds, Nectaries
and Nectar. Springer-Verlag, Dordrecht, The Netherlands, pp 343–376
Petrásek J, Mravec J, Bouchard R, Blakeslee JJ, Abas M, Seifertová D,
Wisniewska J, Tadele Z, Kubes M, Covanová M, et al (2006) PIN
proteins perform a rate-limiting function in cellular auxin efflux. Science
312: 914–918
Poulsen LR, López-Marqués RL, McDowell SC, Okkeri J, Licht D, Schulz A,
Pomorski T, Harper JF, Palmgren MG (2008) The Arabidopsis P4-ATPase
ALA3 localizes to the Golgi and requires a b-subunit to function in lipid
translocation and secretory vesicle formation. Plant Cell 20: 658–676
Radhika V, Kost C, Bartram S, Heil M, Boland W (2008) Testing the optimal defence hypothesis for two indirect defences: extrafloral nectar
and volatile organic compounds. Planta 228: 449–457
Radhika V, Kost C, Boland W, Heil M (2010) The role of jasmonates in
floral nectar secretion. PLoS ONE 5: e9265
Ralph S, Oddy C, Cooper D, Yueh H, Jancsik S, Kolosova N, Philippe RN,
Aeschliman D, White R, Huber D, et al (2006) Genomics of hybrid
poplar (Populus trichocarpa 3 deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of
insect-induced defences in poplar. Mol Ecol 15: 1275–1297
Robinson DG, Jiang L, Schumacher K (2008) The endosomal system of
plants: charting new and familiar territories. Plant Physiol 147: 1482–
1492
Roitsch T (1999) Source-sink regulation by sugar and stress. Curr Opin
Plant Biol 2: 198–206
Rudgers JA, Strauss SY (2004) A selection mosaic in the facultative mutualism between ants and wild cotton. Proc Biol Sci 271: 2481–2488
Ruhlmann JM, Kram BW, Carter CJ (2010) CELL WALL INVERTASE 4 is
required for nectar production in Arabidopsis. J Exp Bot 61: 395–404
Sauer N, Baier K, Gahrtz M, Stadler R, Stolz J, Truernit E (1994) Sugar
transport across the plasma membranes of higher plants. Plant Mol Biol
26: 1671–1679
Schüler G, Goerls H, Boland W (2001) 6-Substituted indanoyl isoleucine
conjugates mimic the biological activity of coronatine. Eur J Org Chem
1663–1668
Sha B, Phillips SE, Bankaitis VA, Luo M (1998) Crystal structure of the
Saccharomyces cerevisiae phosphatidylinositol-transfer protein. Nature
391: 506–510
Shiraishi A, Kuwabara M (1970) The effects of amino acids on the labellar
hair chemosensory cells of the fly. J Gen Physiol 56: 768–782
1190
Plant Physiol. Vol. 159, 2012
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Poplar Extrafloral Nectaries
Smyth GK (2004) Linear models and empirical Bayes methods for assessing
differential expression in microarray experiments. Stat Appl Genet Mol
Biol 3: Article3
Sonwa MM, Kost C, Biedermann A, Wegener R, Schulz S, Boland W
(2007) Dehydrogenation of ocimene by active carbon: artefact formation
during headspace sampling from leaves of Phaseolus lunatus. Arkivoc
2007: 164–172
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for
electron microscopy. J Ultrastruct Res 26: 31–43
Steidle JLM, van Loon JJA (2003) Dietary specialization and infochemical
use in carnivorous arthropods: testing a concept. Entomol Exp Appl 108:
133–148
Stpiczy
nska M, Davies KL, Gregg A (2004) Nectary structure and nectar
secretion in Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge (Orchidaceae). Ann Bot (Lond) 93: 87–95
Stpiczy
nska M, Davies KL, Gregg A (2005) Comparative account of nectary structure in Hexisea imbricata (Lindl.) Rchb. f. (Orchidaceae). Ann
Bot (Lond) 95: 749–756
Svoboda J, Boland W (2010) Plant defense elicitors: analogues of
jasmonoyl-isoleucine conjugate. Phytochemistry 71: 1445–1449
Szyroki A, Ivashikina N, Dietrich P, Roelfsema MR, Ache P, Reintanz B,
Deeken R, Godde M, Felle H, Steinmeyer R, et al (2001) KAT1 is not
essential for stomatal opening. Proc Natl Acad Sci USA 98: 2917–2921
Tautz J (1977) Reception of medium vibration by thoracal hairs of caterpillars of Barathra brassicae L (Lepidoptera, Noctuidae). I. Mechanical
properties of receptor hairs. J Comp Physiol 118: 13–31
Tautz J, Markl H (1978) Caterpillars detect flying wasps by hairs sensitive
to airborne vibration. Behav Ecol Sociobiol 4: 101–110
Tautz J, Rostas M (2008) Honeybee buzz attenuates plant damage by caterpillars. Curr Biol 18: 1125–1126
Trelease W (1881) The foliar nectar glands of Populus. Bot Gaz 6: 284–290
Turner JG (2007) Stress responses: JAZ players deliver fusion and rhythm.
Curr Biol 17: R847–R849
Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U,
Putnam N, Ralph S, Rombauts S, Salamov A, et al (2006) The genome
of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:
1596–1604
Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt M (2009)
A guide to using MapMan to visualize and compare omics data in
plants: a case study in the crop species, maize. Plant Cell Environ 32:
1211–1229
Vassilyev AE (2010) On the mechanisms of nectar secretion: revisited. Ann
Bot (Lond) 105: 349–354
Vesprini JL, Nepi M, Ciampolini F, Pacini E (2008) Holocrine secretion
and cytoplasmic content of Helleborus foetidus L. (Ranunculaceae) nectar.
Plant Biol (Stuttg) 10: 268–271
Wacht S, Lunau K, Hansen K (2000) Chemosensory control of pollen ingestion in the hoverfly Eristalis tenax by labellar taste hairs. J Comp
Physiol A Neuroethol Sens Neural Behav Physiol 186: 193–203
Wenzler M, Hölscher D, Oerther T, Schneider B (2008) Nectar formation and
floral nectary anatomy of Anigozanthos flavidus: a combined magnetic resonance imaging and spectroscopy study. J Exp Bot 59: 3425–3434
Wise RP, Caldo RA, Hong L, Shen L, Cannon E, Dickerson JA (2007)
BarleyBase/PLEXdb. Methods Mol Biol 406: 347–363
Wist TJ, Davis AR (2006) Floral nectar production and nectary anatomy
and ultrastructure of Echinacea purpurea (Asteraceae). Ann Bot (Lond)
97: 177–193
Wooley SC, Donaldson JR, Stevens MT, Gusse AC, Lindroth RL (2007) Extrafloral nectaries in aspen (Populus tremuloides): heritable genetic variation
and herbivore-induced expression. Ann Bot (Lond) 100: 1337–1346
Zárský V, Cvrcková F, Potocký M, Hála M (2009) Exocytosis and cell polarity in
plants: exocyst and recycling domains. New Phytol 183: 255–272
Plant Physiol. Vol. 159, 2012
1191
Downloaded from on July 12, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.